Project Summary The nervous system is precisely wired, and this precision underlies the structure of the neural circuits, and their function. In the central nervous system of vertebrates, and in neuropils of invertebrates, unmyelinated axons are organized into fascicles (or bundles) that form en passant (along the length of the axon) synapses with conjoining axons. The organization of the fascicle limits the contact sites that a given axon makes with potential postsynaptic partners, thereby restricting the position and identity of en passant synapses between neurons. How fascicles are organized in vivo to result in precisely organized neuropils and circuits is a fundamental question in developmental neuroscience. Key fasciculation proteins such as FasII/N-CAM and Cadherin have been identified, but the specific mechanisms that regulate and instruct fascicle formation are unclear. Furthermore, it is unclear how fascicle organization results in accurately formed neuronal circuits and neuropils. This gap remains in part because it has been challenging to examine the dynamic development and organization of fascicles in vivo at single neuron resolution. In this proposal, I will address this knowledge gap by studying the development of the Caenorhabditis elegans nerve ring (the nematode brain). The nerve ring is a neuropil consisting of 185 fasciculating neurons that are precisely positioned during embryonic development. To image the development of the nerve ring we custom-designed a light-sheet microscope capable of continuously imaging the entire 8-hour development of the C. elegans nerve ring at sub-cellular resolution. Combing the genetic and cell biological tools available in C. elegans with our enabling light-sheet microscope, I will identify the cellular and molecular mechanisms that organize fascicle development in vivo to specify the precise formation of neuronal circuits. Neurodevelopmental genes formerly identified in C. elegans are conserved across evolution. Therefore, I foresee that this work will reveal conserved mechanisms of fascicle development in metazoans that could be biomedically relevant for understanding the underpinnings of neurodevelopmental diseases.